Immunophenotyping of Serous Carcinoma of the Female Genital Tract

Immunophenotyping of serous carcinoma of the female genital tract

Sharon Nofech-Mozes1, Mahmoud A Khalifa1, Nadia Ismiil1, Reda S Saad1, Wedad M Hanna1, Al Covens2 and Zeina Ghorab1

1Department of Anatomic Pathology, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, ON, Canada and 2Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Sunnybrook-Odette Cancer Centre, ON, Canada

To update the data on the expression of ‘mesothelioma markers’ by serous carcinomas of various sites we have studied cases from ovary (n ¼ 56), endometrium (n ¼ 37), fallopian tube (n ¼ 6), primary peritoneum (n ¼ 5) and cervix (n ¼ 3) using a panel of antibodies (WT1, P53, estrogen receptors, HER2/neu, D2-40, cytokeratin 5/6 and E-cadherin). Ovarian carcinomas demonstrated D2-40 and cytokeratin 5/6 immunoreactivity in 23.2 and 55.4% of cases, respectively. Endometrial carcinomas demonstrated D2-40 and cytokeratin 5/6 immunoreactivity in 43.2 and 37.8% of cases, respectively. D2-40 staining pattern was predominantly focal; however, strong reactivity was identified in 16.2% of endometrial and 10.7% of ovarian carcinomas. HER2/neu oncoprotein overexpression was demonstrated in 7 of 37 (18.9%) uterine serous carcinomas. In contrast, all the serous carcinomas of the other sites were HER2/neu negative. The proportion of positive cases was significantly different in ovarian vs endometrial carcinomas regarding WT1 (P ¼ 0.0458), estrogen receptors (Po0.001) reactivity and HER2/neu overexpression (P ¼ 0.0025). D2-40 and cytokeratin 5/6 are expressed in a considerable proportion of serous carcinomas and should be used cautiously in a ‘mesothelioma panel’ in situations where serous carcinoma is in the differential diagnosis. HER2/neu was exclusively overexpressed in serous carcinomas of endometrial origin. Modern Pathology (2008) 21, 1147–1155; doi:10.1038/modpathol.2008.108; published online 20 June 2008

Keywords: serous carcinoma; primary peritoneal carcinoma; D2-40; CK 5/6; HER2/neu; ER

Serous carcinoma is an aggressive type of cancer the tumor exhibits similar morphologic features, that may arise from various organs of Mullerian characterized by the presence of cellular papillae, lineage, including ovaries, endometrium, fallopian slit-like glandular spaces, small detached buds and tubes, peritoneum and rarely the cervix. Serous tufts, moderate to severe cytologic atypia, frequent carcinoma accounts for about half of the malignant mitotic activity, necrosis and occasional concentric ovarian tumors and about 10% of endometrial calcification (psammoma bodies). The histologic cancers. Despite intense investigations, ovarian distinction between serous carcinomas of various serous carcinoma and uterine serous carcinoma origins, most commonly the ovary vs endometrium, are associated with poor prognosis. Patients with may be difficult especially in cases with advanced ovarian serous carcinoma most commonly present disease that involves more than one site in the with stage III tumors and their 5-year survival is female genital tract or when it diffusely involves the 20%.1,2 Uterine serous carcinoma is notorious for its peritoneum. As uterine serous carcinoma frequently propensity to invade the myometrium and lympho- involves the ovaries, in cases with dual organ vascular channels and is associated with a 5-year involvement it may be difficult to ascertain the survival of 36%. This is attributable to the presence primary site. Although gene expression profiling of extrauterine disease at the time of diagnosis in may differentiate between tumors of ovarian and the majority of cases.1,2 Regardless of its origin, endometrial origin,3 these tumors share some molecular abnormalities such as in p53 or p16.4 As a result, there is no known marker that is absolutely Correspondence: Dr S Nofech-Mozes, MD, Department of Anatomic specific for a particular site. However, a panel of Pathology, Sunnybrook Health Sciences Centre, University of antibodies may facilitate identifying the site of Toronto, 2075 Bayview Avenue E4-23a, Toronto, ON, Canada origin. This panel traditionally includes WT1 and M4N 3M5. 5–8 E-mail: [email protected] P53. Accurate identification whether a serous Received 10 March 2008; revised 16 May 2008; accepted 18 May carcinoma originates from the ovary or the endome- 2008; published online 20 June 2008 trium is clinically important as the pattern of tumor Immunohistochemistry of serous carcinoma S Nofech-Mozes et al 1148 spread and the adjuvant therapy may differ, the Materials and methods latter is thought to exhibit a poorer response to platinum-based compounds.9 Ovarian serous carci- Cases noma characteristically spread intraperitoneally We studied 107 consecutive cases of serous carci- (transcelomic); whereas uterine serous carcinoma noma of the ovary, endometrium, fallopian tube, spread by both transcelomic and lymphovascular primary peritoneum and cervix accessioned during invasion. In addition, uterine serous carcinoma the period of 1999–2007 in the Department of commonly involves the ovaries whereas ovarian Anatomic Pathology, Sunnybrook Health Sciences serous carcinoma rarely spread to the endometrium Centre in Toronto. Pertinent demographic and and is more likely to involve the uterine serosa. clinical data were retrieved from the electronic Immunohistochemical support for a site of origin medical records. would be helpful in cases featuring a superficially Cases of mixed cell type were excluded from the myoinvasive uterine serous carcinoma and coex- study. Ovarian serous carcinomas were included istent ovarian, omental or peritoneal disease. In only when destructive stromal invasion was identi- such cases the question arises as to whether these fied. Borderline serous tumors with or without represent synchronous neoplasms or one tumor microinvasion were excluded. Ovarian serous carci- with a metastatic spread. Immunohistochemistry nomas were graded using the Silverberg’s scoring may also be useful when surgical resection is system16 and all uterine serous carcinomas were following neoadjuvant chemotherapy or when lim- considered as high grade. We acknowledge that ited diagnostic material is available, ie, cell blocks neoadjuvant chemotherapy can induce morphologic from malignant ascites, biopsies of metastatic carci- changes that could potentially alter the ovarian noma or endometrial biopsies. Another challenge is cancer grade. The diagnosis of primary peritoneal to differentiate ovarian serous carcinoma from meso- carcinoma was made only in the absence of thelial cells particularly when limited diagnostic concurrent or previous primary endometrial, ovar- material is provided such as cytology preparation. ian or fallopian tube carcinoma, or in the presence of Commonly in these cases, serous carcinoma cells microscopic foci of tumor on the external surface of should be distinguished from benign mesothelial the ovaries less than 5 mm. The diagnosis of cells that may form tight clusters that mimic ovarian fallopian tube carcinoma was made when the tumors or less frequently from epithelioid peritoneal fallopian tube contained the main bulk of the tumor mesothelioma. Recently introduced immunohisto- whereas the adjacent ovary had a minor component. chemical markers such as D2-40, CK 5/6, E-cadherin The diagnosis of cervical serous carcinoma was and HER2/neu (SP3) have been tested only on a made only in the absence of concurrent or previous limited number of cases of serous carcinoma. The primary endometrial, ovarian or peritoneal serous proportion of positive tumors and staining pattern of carcinoma as previously described.17 these markers in serous tumors of different sites within the female genital tract is unknown. A small number of reports suggest that ovarian serous Immunohistochemistry tumors express ‘mesothelioma markers’.10–12 D2-40, a marker of lymphatic endothelium as well as Two gynecologic pathologists reviewed the cases benign and malignant mesothelial cells, and a and selected a block containing representa- specific high molecular cytokeratin (CK) 5/6 were tive tumor for immunohistochemical studies. considered as useful ‘mesothelioma markers’.12–15 These were performed on formalin-fixed paraffin- The aim of this study is to characterize the embedded tissue sections using a panel of antibodies expression ‘mesothelioma markers’ and a panel of raised against: WT1, p53, estrogen receptors (ER), biological markers by serous tumors of the female HER2/neu (SP3), D2-40, CK 5/6 and E-cadherin. The genital tract and to explore possible variation in primary antibodies that were used are listed in Table 1. expression of markers among different sites. WT1 and p53 were included in this study mainly as

Table 1 Antibodies used in the study

Antibody Manufacturer Dilution Pretreatment Detection

WT1 Dako M3561 1:100 HIER citrate (pH 6.0) Biocare Mach 3 Polymer P53 (DO7) NovoCastra NCL-p53-DO7 1:500 HIER citrate (pH 6.0) Biocare Mach 3 Polymer ER NovoCastra NCL-ER-6F11 1:200 HIER citrate (pH 6.0) Biocare Mach 3 Polymer HER2/neu (SP3) NeoMarker RM-9103 1:200 HIER citrate (pH 6.0) Biocare Mach 4 Polymer D2-40 Research laba 1:20 000 No pretreatment Biocare Mach 3 Polymer CK 5/6 Dako M7237 1:400 HIER Borg (pH 9.5) Biocare Mach 3 Polymer E-cadherin BD Biosciences, city and state for all of them 1:2000 HIER citrate (pH 6.0) ZyMed LSAB detection kit

CK, cytokeratin; ER, estrogen receptor; lab, laboratory. aA generous gift from Dr Marks, Women’s College Hospital Toronto, Canada.37,38

Modern Pathology (2008) 21, 1147–1155 Immunohistochemistry of serous carcinoma S Nofech-Mozes et al 1149 a complementary tool in making the diagnosis of Table 3 Number of positive cases and the percentage of serous carcinoma. Immunostaining was recorded immunoreactive cells for the markers by site of origin semiquantitatively by estimating the proportion Percentage of of positive cells and classified as: negative ¼ 0%; immunoreactive cells ‘1 þ ’ ¼ 1–25%; ‘2 þ ’ ¼ 25–50% and ‘3 þ ’ ¼strong (450%). HER2/neu was considered positive only 0 1–25% 25–50% 450% when complete strong membrane staining was identified in more than 30% of the tumor, as Ovary (n ¼ 56) outlined in the updated recommendations for WT1 — — — 56 HER2/neu testing in breast cancer.18 P53 4 4 1 47 HER2/neu gene amplification was tested by FISH ER 6 7 7 36 HER2/neu 55 — — — using the Vysis probe (Vysis Inc., Downers Grove, IL, D2-40 43 7 — 6 USA) following the manufacturer’s instructions. In CK 5/6 25 20 3 8 brief, the, PathVysiont kit is based on dual-color E-cadherin 1 — — 55 interphase analysis by using combined Her2/neu Endometrium (n ¼ 37) (rhodamine labeled) and chromosome 17—a-satel- WT1 18 1 — 18 lite DNA probes (CEP17, fluorescein labeled). P53 1 2 3 31 ER 23 7 3 4 HER2/neu 29 1 — 7 D2-40 18 5 5 6 Statistics CK 5/6 23 10 4 — Fisher’s exact test was used to examine differences E-cadherin — — — 37 in proportions of positive stains between ovarian Fallopian tube (n ¼ 6) and endometrial carcinomas. WT1 — — — 6 This study was approved by the Institutional P53 1 — — 5 Research Ethics Board. ER 1 4 1 HER2/neu 6 — — — D2-40 5 1 — — CK 5/6 6 — — — Results E-cadherin — — — 7 Immunohistochemistry was performed on 56 cases Primary peritoneal (n ¼ 5) of ovarian serous carcinomas, 37 uterine serous WT1 — 2 2 1 P53 — — 1 4 carcinomas, 6 fallopian tube serous carcinomas, 5 ER 2 2 1 — primary peritoneal serous carcinomas and 3 endo- HER2/neu 5 — — — cervical serous carcinomas that met our inclusion D2-40 5 — — — criteria. The case characteristics are summarized in CK 5/6 5 — — — Table 2 and the immunohistochemical results are E-cadherin — — — 5 summarized in Table 3. Cervix (n ¼ 3) WT1 2 — — 1 P53 1 — — 2 ER 2 1 — — WT1 HER2/neu 3 — — — D2-40 5 — — — All ovarian serous carcinomas demonstrated WT1 CK 5/6 — — — 3 reactivity in450% of the tumor cells, whereas 18 of E-cadherin — — — 3 37 (48.6%) of uterine serous carcinomas demonstrated a similar degree of reactivity for WT1 CK, cytokeratin; ER, estrogen receptor.

Table 2 Case characteristics

Site of origin Ovary Endometrium Fallopian tube Primary peritoneal Cervical (n ¼ 56) (n ¼ 37) (n ¼ 6) (n ¼ 5) (n ¼ 3)

Age (years) 59.4±13.5 71.1±8.1 56.1±4.1 70.1±9.7 58.5±6.4 Grade 1 9 — 1 1 — Grade 2 7 — — 1 — Grade 3 35 37 5 3 3 Unspecifieda 5— — — — Neoadjuvant chemotherapy 30 — 2 — — aNeoadjuvant chemotherapy-related changes preclude accurate grading.

Modern Pathology (2008) 21, 1147–1155 Immunohistochemistry of serous carcinoma S Nofech-Mozes et al 1150 (P ¼ 0.0458). In all fallopian tube carcinomas, 1 of 5 uterine serous carcinomas were ER negative. (20%) primary peritoneal carcinomas and 1 of 3 Only 4 of 37 (10.8%) demonstrated ER reactivity cervical serous carcinomas 450% of the tumor were in more than 50% of the tumor (Po0.001). WT1 immunoreactive. Similar degree of ER reactivity was demonstrated only in 1 of 6 (16.7%) fallopian tube tumors and in none of the primary peritoneal or cervix serous P53 carcinomas. The proportion of strong p53 reactivity was compar- able in all disease sites. p53 was demonstrated in more450% of the tumor in 47 of 56 (83.6%) ovarian HER2/neu serous carcinomas, 28 of 34 (83.8%) uterine serous carcinomas, 5 of 6 (83.9%) fallopian tube, 4 of 5 Strong complete membranous stain consistent with (80%) primary peritoneal carcinomas and in 2 of 3 HER2/neu oncoprotein overexpression was demons- cervical serous carcinomas. trated in 7 of 37 (18.9%) uterine serous carcinomas (Figure 1). Notably, one additional uterine serous carcinoma was only focally HER2/neu positive. This Estrogen Receptors case was further studied by FISH that showed focal HER2/neu gene amplification. The FISH score in the Estrogen receptor reactivity was demonstrated in IHC-positive area was 3 whereas that of the IHC- more than 50% of the tumor of 36 of 56 (64.3%) negative area was 1. None of the other sites ovarian serous carcinomas, whereas most of the including ovary, fallopian tubes, primary peritoneal

Figure 1 (a) Hematoxylin–eosin, uterine serous carcinoma. (b) HER2/neu oncoprotein (SP3) is overexpressed. Note complete strong membranous staining. (c) Hematoxylin–eosin, uterine serous carcinoma and adjacent benign endometrial gland. (d) HER2/neu oncoprotein (SP3) is overexpressed in the tumor, the benign endometrial gland is negative.

Modern Pathology (2008) 21, 1147–1155 Immunohistochemistry of serous carcinoma S Nofech-Mozes et al 1151

Figure 2 Ovarian serous carcinoma: D2-40 immunohistochemistry. (a) Positive staining along the apical cell membrane. (b) Patient treated with neoadjuvant chemotherapy with similar staining pattern. Uterine serous carcinoma: D2-40 immunohistochemistry. (c) Continuous-positive staining along the apical cell membrane. Note D2-40 is also positive in stromal lymphatics. The benign adjacent endometrial gland is negative. (d) Uterine serous carcinoma, similar staining pattern. and cervix tumors overexpressed HER2/neu onco- occasional cases exhibited continuous membranous protein. The difference in the proportion of positive stain encircling the cells. cases between endometrial and ovarian tumors was significant (P ¼ 0.0025). CK 5/6

D2-40 Some degree of CK 5/6 reactivity was demonstrated in 31 of 56 (55.4%) ovarian serous carcinomas but Some degree of D2-40 reactivity was demonstrated 450% of the tumor was reactive only in 8 of 56 in 13 of 56 (23.2%) ovarian serous carcinomas, but (14.3%; Figure 3). Some degree of CK 5/6 reactivity only in 6 of 56 (10.7%) cases 450% of the tumor was demonstrated in 14 of 37 (37.8%) uterine serous was positive (Figure 2a and b). Some degree of D2-40 carcinomas (Figure 4). None of the uterine serous reactivity was seen in 16 of 37 (43.2%) uterine carcinomas, fallopian tube carcinomas and primary serous carcinomas but only in 6 of 37 (16.2%) cases peritoneal carcinomas reacted with CK 5/6 in 450% 450% of the tumor was positive (Figure 2c and d). of the tumor. The three cases of cervical serous The difference between D2-40 expression in ovarian carcinoma demonstrated CK 5/6 reactivity in 450% vs endometrial tumors was not statistically signifi- of the tumor. cant (P ¼ 0.198). Positive cases from other sites The difference between CK 5/6 expression in demonstrated less than 50% of the tumor cells were ovarian vs endometrial tumors was not statistically immunoreactive. Characteristically, the staining significant (P ¼ 0.349). In ovarian and uterine serous pattern was along the apical cell membrane though carcinomas that were focally reactive for both

Modern Pathology (2008) 21, 1147–1155 Immunohistochemistry of serous carcinoma S Nofech-Mozes et al 1152

Figure 3 (a) Ovarian serous carcinoma; hematoxylin–eosin. (b) A representative case of ovarian serous carcinoma with strong and diffuse CK 5/6 immunoreaction. (c) Ovarian serous carcinoma; hematoxylin–eosin. (d) A representative case of ovarian serous carcinoma with CK 5/6 immunoreaction in less than 25% of the tumor cells. This was the predominant pattern.

Figure 4 (a) Uterine serous carcinoma; hematoxylin–eosin. (b) CK 5/6-positive immunoreaction is demonstrated in less than 25% of the tumor cells.

Modern Pathology (2008) 21, 1147–1155 Immunohistochemistry of serous carcinoma S Nofech-Mozes et al 1153 CK 5/6 and D2-40, the subset of CK 5/6-positive cells the immunohistochemical profile of ovarian serous did not overlap with D2-40-positive cells. carcinoma with epithelioid mesotheliomas using a panel of 15 markers. In their study, none of the ovarian serous carcinoma reacted strongly with E-cadherin D2-40 and CK 5/6; however, 13% exhibited focal D2-40 reactivity. Chu et al noted that 17 of 26 (65%) Regardless of their site of origin, almost all serous of the ovarian serous carcinoma included in their carcinomas that were included in the study exhibited study, exhibited focal D2-40 reactivity. The differ- diffuse E-cadherin reactivity. One ovarian serous ence in proportion of D2-40 and CK 5/6-positive carcinoma was negative in repeated staining with a cases was not significant when ovarian serous positive internal control. carcinoma were compared with uterine serous carcinoma in our study. Ionescu et al25 suggested that Effect of Neoadjuvant Therapy CK 5/6 expression is significantly higher in uterine serous carcinoma based on a TMA study. They found In 30 of 56 (53.6%) of the ovarian serous carcinoma that CK 5/6 was expressed in 48% of uterine serous patients, neoadjuvant chemotherapy was adminis- carcinoma (compared to 37.8% in our study) but only tered prior to the primary surgical treatment. There in 5% of ovarian serous carcinoma (compared to was no significant association between neoadjuvant 56.4% in our study). Their observation regarding therapy and expression of any of the markers. HER2/neu oncoprotein overexpression in uterine serous carcinoma is in line with our findings; however, their cut off for positivity was not specified. Association with Tumor Grade As shown by an oligonucleotide microarray study assessing the differential expression of approxi- When low grade (Silverberg’s grade 1) was com- mately 10 000 genes between ovarian serous carci- pared with high-grade (Silverberg’s grades 2 and 3) noma and uterine serous carcinoma, HER-2/neu is ovarian serous carcinomas, there was no significant the most strikingly overexpressed gene in uterine association between the ovarian tumor grade and the serous papillary carcinoma compared with serous expression of any of the markers. epithelial ovarian cancer.26,27 It has been shown that HER-2/neu oncoprotein overexpression is signifi- Discussion cantly associated with poor survival in uterine serous carcinoma28 and could also be important as D2-40 antibody reacts with a mucin-type transmem- a predictive factor in uterine serous carcinoma brane sialoglycoprotein19,20 expressed in lymphatic treatment. It has been related to resistance to endothelium, seminoma, benign and malignant chemotherapy and poor prognosis in patients with mesothelial cells.15,20,21 In the female genital tract this aggressive uterine malignancy.29 Trastuzumab, a D2-40 is expressed in germ cell neoplasia, sex cord humanized monoclonal antibody specific for HER-2/ stromal tumors and adenomatoid tumors.19,22 Pre- neu, is important in the adjuvant and metastatic vious studies suggested that D2-40 and CK 5/6 are treatment of patients with HER-2/neu-overexpres- specific to mesothelium and recommended their use sing breast cancer. HER-2/neu overexpression in to differentiate mesothelioma from serous carcinoma uterine serous carcinoma provides a biological of the ovary.13,23,24 In our study, D2-40 and CK 5/6 rationale for the use of trastuzumab in the treatment expression was demonstrated in a substantial of this malignancy.27,30,31 A novel rabbit monoclonal number of serous carcinoma of ovarian (23.2 and antibody directed against HER2/neu oncoprotein 55.4%, respectively) and endometrial origin (43.2 SP3 was recently introduced and is used in routine and 37.8%, respectively). Nevertheless, unlike the clinical diagnosis in our laboratory. We have shown diffuse reactivity characteristic to mesothelioma, the that using this novel antibody for HER2/neu testing expression of D2-40 and/or CK 5/6 in serous reduces the rate of equivocal immunohistochemical carcinomas was usually confined to a subset of the results and highly correlates with HER2/neu gene neoplastic cells; typically the stain was positive in amplification.32 In endometrial cancer, HER2/neu less than 25% of the cells. Our findings confirm oncoprotein overexpression has been associated results of a previous study that demonstrated low with type II endometrial cancer, namely uterine specificity for D2-40 as a mesothelial marker, serous carcinoma. An earlier study26 on HER2/neu especially in the context of differentiating mesothe- status in uterine serous carcinoma reported that lial cells from an ovarian carcinoma. Unlike our 35% of 26 cases examined overexpressed HER2/neu study, the aforementioned study examined D2-40 oncoprotein and demonstrated gene amplification expression in effusion specimens and in multi- in all the cases scored 3 þ by immunohistochem- tissue microarray that included only 23 cases of istry. Interestingly, they also noted cases with primary or metastatic unspecified ovarian carcino- intratumoral heterogeneity. mas. The authors reported that 19% of the cores and Our observation that ER is expressed in almost all more than 50% of effusions of ovarian carcinomas (89%) ovarian serous carcinomas is in line with were positive for D2-40.11 Ordo´n˜ ez et al compared previous studies. Ordo´n˜ ez33 reported ER expression

Modern Pathology (2008) 21, 1147–1155 Immunohistochemistry of serous carcinoma S Nofech-Mozes et al 1154 in 88% of ovarian serous carcinomas (n ¼ 40), with United States and Canadian Academy of Pathology similar frequency in primary peritoneal carcinomas, Annual Meeting, Denver, Colorado, March 2008. and Comin et al34 found that 95% of ovarian serous carcinomas included in their study were ER positive. The expression of ER in ovarian serous carcinomas in our study was not associated with References tumor grade or neoadjuvant chemotherapy. In contrast to our observation, higher ER and E-cadherin 1 Crum CP, Lee KR. The pathology of surface epithelial- expression was reported for low-grade ovarian stromal tumors of the ovary. Diagnostic Gynecologic and Obstetric Pathology. Elsevier Saunders: Philadel- serous carcinomas than for high-grade ovarian 35 phia, PA, USA, 2006, pp 839–903. serous carcinomas by Wong et al. It is possible 2 Kurman RJ. Blaustein’s Pathology of the Female that we could not detect a significant difference in Genital Tract, 5th edn. Springer-Verlag: Baltimore, ER expression between low- and high-grade ovarian MD, 2001. serous carcinoma in any of the markers results from 3 Zorn KK, Bonome T, Gangi L, et al. Gene expression limited sample size of low-grade tumors in our profiles of serous, endometrioid, and clear cell sub- study. O’Neill et al36 found significantly higher types of ovarian and endometrial cancer. Clin Cancer expression of p53 and HER2/neu in high-grade Res 2005;11:6422–6430. ovarian serous carcinoma when compared with 4 Chiesa-Vottero AG, Malpica A, Deavers MT, et al. low-grade tumors. The authors used a different cut Immunohistochemical overexpression of p16 and p53 off in their statistical analysis as well as a different in uterine serous carcinoma and ovarian high-grade antibody for HER2/neu testing that may account for serous carcinoma. Int J Gynecol Pathol 2007;26: 328–333. the difference between our findings and their report. 5 Al-Hussaini M, Stockman A, Foster H, et al. WT-1 In accordance with our observation, expression of assists in distinguishing ovarian from uterine serous WT1 by serous carcinoma other then ovarian origin carcinoma and in distinguishing between serous and has been previously demonstrated in fallopian tube endometrioid ovarian carcinoma. Histopathology and primary peritoneal but not in uterine serous 2004;44:109–115. carcinomas.8 Acs et al7 found that a subset of uterine 6 Goldstein NS, Uzieblo A. WT1 immunoreactivity in serous carcinoma (10 of 16) express WT1; however, uterine papillary serous carcinomas is different from the level of WT1 expression in their study was ovarian serous carcinomas. Am J Clin Pathol 2002;117: significantly different among serous carcinomas aris- 541–545. ing at different sites (Po0.0001). Similar to our 7 Acs G, Pasha T, Zhang PJ. WT1 is differentially findings the authors did not find a significant expressed in serous, endometrioid, clear cell, and mucinous carcinomas of the peritoneum, fallopian difference in p53 expression among the different sites. tube, ovary, and endometrium. Int J Gynecol Pathol In summary, using a panel of seven antibodies, we 2004;23:110–118. found a significant difference in immunoreactivity 8 Hashi A, Yuminamochi T, Murata S, et al. Wilms tumor of ovarian and endometrial carcinomas regarding gene immunoreactivity in primary serous carcinomas WT1 (P ¼ 0.0458) and ER (Po0.001) reactivity as of the fallopian tube, ovary, endometrium, and perito- well as HER2/neu oncoprotein overexpression neum. Int J Gynecol Pathol 2003;22:374–377. (P ¼ 0.0025). Strong WT1 and ER expression were 9 Resnik E, Taxy JB. Neoadjuvant chemotherapy in associated with ovarian primary whereas HER2/neu uterine papillary serous carcinoma. Gynecol Oncol oncoprotein overexpression was associated with 1996;62:123–127. endometrial origin. 10 Jones DL, Denning KL, Saad RS, et al. Diagnostic D2-40 and CK 5/6 are expressed in considerable limitation of D2-40 in separating serous carcinoma proportion of serous carcinomas and should be used from other metastatic carcinoma of the abdomen [abstract]. Mod Pathol 2006;19:183A. cautiously in a ‘mesothelioma panel’ in particular 11 Bassarova AV, Nesland JM, Davidson B. D2-40 is not a when used to differentiate benign reactive mesothelial specific marker for cells of mesothelial origin in serous proliferation or epithelioid mesothelioma from serous effusions. Am J Surg Pathol 2006;30:878–882. carcinoma in fluid cytology. The focal CK 5/6 and 12 Chu AY, Litzky LA, Pasha TL, et al. Utility of D2-40, a D2-40 expression in substantial number of cases may novel mesothelial marker, in the diagnosis of malig- limit the value of microarray studies. HER2/neu was nant mesothelioma. Mod Pathol 2005;18:105–110. exclusively overexpressed in serous carcinomas of 13 Ordo´n˜ ez NG. The diagnostic utility of immunohisto- endometrial origin and although expressed in limited chemistry and electron microscopy in distinguishing subset of cases, could be used to identify endometrial between peritoneal mesotheliomas and serous carci- origin in the context of advanced serous carcinomas, nomas: a comparative study. Mod Pathol 2006;19: and may be used to select patients for targeted therapy. 34–48. 14 Ordo´n˜ ez NG. D2-40 and podoplanin are highly specific and sensitive immunohistochemical markers of epithelioid malignant mesothelioma. Hum Pathol 2005;36: Acknowledgement 372–380. 15 Bhalla R, Siddiqui MT, Mandich D, et al. Diagnostic We thank Kevin Kowk for his skillful technical utility of D2-40 and podoplanin in effusion cell blocks. assistance. This study was presented in part in the Diagn Cytopathol 2007;35:342–347.

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